Summary/Abstract Cardiometabolic disorders, which have emerged as major health problems on a global scale, share a number of common risk factors (e.g., obesity, hypertension, dyslipidemia, insulin resistance, inflammation, etc.), which likely interact with genetic and epigenetic factors. Genome-wide association studies (GWAS) have identified hundreds of common variants associated with these complex traits; however, they explain only a small fraction of the phenotypic variance. Recently, increased attention is being paid to the role of epigenetic variation in the etiology of complex diseases. Uncovering the underlying genetic and epigenetic factors that contribute to the risk for cardiometabolic disorders, as well as their interaction with health behaviors and dietary factors, is significant in that it may lead to newer strategies for prevention, treatment, and management. We propose a comprehensive epigenome-wide association study (EWAS) of cardiometabolic traits using longitudinal samples obtained at two discrete time-points 9-years apart, and integrate it with data from a previously conducted GWAS. We hypothesize that (1) ?stable? epigenetic marks, with limited intra-individual variation established in early life, define developmental trajectory of cardiometabolic traits; (2) ?dynamic? marks that fluctuate over life- course in response to endogenous biological processes and exogenous environmental stressors may also contribute to development of cardiometabolic disorders later in life; and (3) these epigenetic marks interact with genetic variation to influence phenotypic variability. To test these hypotheses, we will leverage an existing resource of >1,400 samples and data (phenotypic, environmental, DNA, GWA) collected in 2007-08 in our prior GWAS of metabolic syndrome in a relatively isolated population from the eastern Adriatic coast of Croatia. We will follow-up >1,000 of the individuals sampled in the prior study 9 years later, with collection of blood samples, demographic, behavioral, environmental, and phenotypic data (Aim 1); conduct an EWAS, applying the Infinium 450K methylation array to paired blood samples from each of the two discrete time points to assess methylation patterns while adjusting for inter-personal variability in relative leukocyte fractions using a recently developed method for cellular deconvolution (Aim 2); and conduct an integrative genome- and epigenome- wide analysis to examine their joint effects on trait variability using a bioinformatic and systems biology approach (Aim 3). Such a study is necessary to better elucidate the role of epigenetics in cardiometabolic disorders, and the potential interplay with genetic factors, providing new avenues for prevention, treatment, and management strategies to help alleviate the collective global impact of this devastating group of diseases. The project is innovative in its (1) use of a unique population cohort; (2) adjustment of cellular heterogeneity of peripheral blood samples; (3) defining the roles of ?stable? and ?dynamic? epigenetic marks; and (4) it will be among the first large-scale integrative EWAS and GWAS of cardiometabolic traits that will have broader impact on design and conduct of genetic and epigenetic epidemiological studies of complex traits.